The development of chemical sensors began in 1906 when it was discovered that the electrochemical potential at certain thin glass membranes depended upon the hydrogen ion concentration in the bathing solution (1). As a result of this discovery, the first pH electrode was reported in 1909 (2). Since that time, development of chemical sensors has been considerable with blood gas and electrolyte measurement systems having undergone substantial development. In comparison, development of sensors for the detection of metabolites and biomolecules has not progressed as far.
The term “biosensor” usually denotes a sensor which is specific for biological substances. However, the term is often used to specify a sensor which uses biological substances to detect other chemicals. A biosensor is a monitoring device whose selectivity in detecting an analyte is the result of the binding specificity of a biological molecule, e.g., antibody, enzyme or membrane receptor. Analyte concentrations are determined by “transducing” these analyte binding events into a measurable quantity such as an electronic or optical signal. Thus, the basic components of a biosensor are a biological molecule, e.g., antibody, enzyme or membrane receptor, and a transducer.
Biosensors generally fall into three basic categories: electrochemical; optical; or physical. These biosensors incorporate transducers which are well known to the skilled artisan and include calorimetric, piezoelectric, amperometric, optical fiber, optical waveguide, lipid membrane, potentiometric and electrochemical capacitance/impedance devices.
While the aforementioned biosensor transduction techniques and devices may be employed in the invention, future improvements in miniaturization and in other analytical techniques such as mass spectroscopy, gas chromatography and nuclear magnetic resonance spectroscopy may allow such other techniques and systems to also be used in the invention.
The biological component of preexisting biosensors is either an enzyme, an antibody, a membrane receptor, whole cell or tissue. Enzymes, antibodies and membrane receptors are all biological macromolecules whose function is to bind target molecules in a highly specific manner. While integration of enzymes and receptors into sensors appeared to have great potential, their commercial application is often limited.
Sensors incorporating enzymes can detect many chemicals. However, the chemicals which can be detected are limited to those for which stable enzymes are available and still further to those enzymes which either consume or produce measurable molecules. Yet a large number of chemicals are consumed or produced by chemical reactions for which there are no known enzyme catalysts. Biosensors which rely upon enzymes are also limited to the extent that the enzymes are limited in their function by allosterism. Allosteric enzymes are enzymes which are stimulated or inhibited by a modulator molecule which may be the substrate, the product or some other molecule. As a result of allosterism, the kinetic behavior of such enzymes is greatly altered by variations in the concentration of the modulator. A relatively simple example of allosteric behavior is where the enzyme is subject to feedback inhibition, i.e., where the catalytic efficiency of the enzyme decreases as the concentration of an intermediate or subsequent product increases. Use of such enzymes in many biosensor applications is thus limited and requires continuous removal of product.
A further major disadvantage of enzyme sensors is attributable to the fact that the binding affinity of the analyte is determined by its biological role. Most enzymes possess binding affinities in the 103-106M−1 range and the binding affinity determines the sensitivity of the enzyme sensor. Thus most enzyme sensors have, at best, nanomolar detection limits.
As to membrane receptors, there is difficulty in isolating sufficient quantities of protein. Moreover, the commercial application of membrane receptors in sensors is often limited by their inherent instability and by the need for highly ordered and easily degraded environments.
Sensors incorporating antibodies as the biological component, like enzymes, have high selectivity but can have substantially improved detection limits as compared to enzymes due to the higher binding affinities of antibodies. Antibodies have binding affinities of 105-1013M−1 and thus sensors incorporating antibodies can have detection limits in the picomolar range. Antibodies are limited, however, by the fact that the recognition and identification of antigens by antibodies via immunological reactions is usually a one-time event. As antigen saturates antibody, the development of a specific antigen-antibody complex can be measured. However, whether monoclonal or polyclonal antibodies are used for antigen capture, this reaction (with high affinity bindings and hence improved detection limits) is usually not readily reversible. The antibody-based sensor thus becomes saturated and the presence of analytes in a subsequent sample cannot be detected. The inability to repeatedly use such sensors is a major limitation.
A common method for dissociating antigen-antibody complexes involves incubating the antibody in either low pH solutions or high concentrations of salt, urea or thiocyanate. While such methods can regenerate an antigen binding site, each procedure requires careful control of the reaction conditions and may cause partial protein denaturation, especially after several regeneration cycles. The absence of a reliable method for regenerating an antigen binding site immediately after each binding event has frustrated the development of a sensing system capable of monitoring successive exposures to a given analyte.
It can be readily appreciated that a durable sensing system with high sensitivity which is capable of repeatedly detecting and measuring the concentration of an analyte over large concentration ranges and which is capable of being calibrated without antigen saturation would be a highly desirable advance over the current state of the art in sensing systems.